Previously, we had discovered N-RAP, a novel, muscle specific protein with homology to nebulin. The C-terminal half of N-RAP contains more than two complete 245 residue nebulin-like super repeats. The N-terminus contains the consensus sequence of a cysteine rich LIM domain, which may function in mediating protein-protein interactions. Our work had localized N-RAP at the myotendon junction in skeletal muscle and at the intercalated disk in cardiac muscle, leading us to hypothesize that N-RAP may be involved in linking the ends of myofibrils to specialized protein complexes found beneath the sarcolemma. We further explored this hypothesis by surveying the ability of recombinant N-RAP domains to bind a variety of candidate proteins. Initial studies suggested that N-RAP domains bind to a wide variety of proteins found in myofibrils and at myofibril ends; however, control experiments indicate that many of these proteins actually adhere to the histidine tag that is present in all of the recombinant N-RAP fragments. Actin is the only myofibrillar protein tested that exhibits specific binding to N-RAP, with high affinity binding to N-RAP super repeats, and ten-fold weaker binding to the region of N-RAP in between the super repeats and the LIM domain. In contrast, tropomyosin, troponin, whole myosin and isolated myosin heads exhibited no specific binding to N-RAP domains. A recombinant fragment corresponding to the C-terminal one fourth of vinculin also binds specifically to N-RAP super repeats, while no specific N-RAP binding activity was observed for other regions of the vinculin molecule. Finally, talin binds with high affinity to the LIM domain of N-RAP. The results are consistent with our original hypothesis that N-RAP may serve as an important mechanical link in transmitting tension from the myofibrils to the extracellular matrix. In the past year, we completed the sequencing of cDNA representing the N-terminal and C-terminal regions of mouse nebulin. Our data show that the N-terminal and C-terminal regions of nebulin are highly conserved between mice and humans. However, the C-terminal region of mouse nebulin is shorter than this region in human nebulins due to the presence of only 16 modules in this region, as compared to 20 and 23 modules in fetal and adult human nebulins, respectively. Comparison of the mouse and human sequence suggests that extensive variations in the number of C-terminal modules may partially account for the developmental, species, and tissue-specific size variations previously observed for the nebulin protein.